The strongly conserved Lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer

Hans Krautwurst, Sergio Bazaes, Fernando D. González, Ana María Jabalquinto, Perry A. Frey, Emilio Cardemil

Research output: Contribution to journalArticlepeer-review

49 Citations (Scopus)

Abstract

Lysine 256, a conserved amino acid of Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered K(m) for MnADP but about a 20 000-fold decrease in V(max) for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyrorate kinase-like activity. The dissociation constant for the enzymeMnATP complex was 1.3 ± 0.3 μM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 ± 0.6 μM, 17 ± 2 μM, and 20 ± 6 μM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.

Original languageEnglish
Pages (from-to)6295-6302
Number of pages8
JournalBiochemistry
Volume37
Issue number18
DOIs
Publication statusPublished - 5 May 1998

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'The strongly conserved Lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer'. Together they form a unique fingerprint.

Cite this